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Infection and Immunity, October 2008, p. 4479-4488, Vol. 76, No. 10
0019-9567/08/$08.00+0     doi:10.1128/IAI.00610-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Adaptation of Francisella tularensis to the Mammalian Environment Is Governed by Cues Which Can Be Mimicked In Vitro{triangledown} ,{dagger}

Karsten R. O. Hazlett,1* Seth D. Caldon,1 Debbie G. McArthur,2 Kerry A. Cirillo,1 Girish S. Kirimanjeswara,1 Micheal L. Magguilli,2 Meenakshi Malik,1 Aaloki Shah,1 Scott Broderick,4 Igor Golovliov,3 Dennis W. Metzger,1 Krishna Rajan,4 Timothy J. Sellati,1 and Daniel J. Loegering2

Center for Immunology and Microbial Disease,1 Center for Cardiovascular Sciences, Albany Medical College, 47 New Scotland Avenue, Albany, New York 12208,2 Department of Bacteriology, Umea University, SE-901 85 Umea, Sweden,3 Department of Material Sciences and Engineering & Bioinformatics and Computational Biology, Iowa State University, Ames, Iowa 500114

Received 17 May 2008/ Returned for modification 30 June 2008/ Accepted 7 July 2008

The intracellular bacterium Francisella tularensis survives in mammals, arthropods, and freshwater amoeba. It was previously established that the conventional media used for in vitro propagation of this microbe do not yield bacteria that mimic those harvested from infected mammals; whether these in vitro-cultivated bacteria resemble arthropod- or amoeba-adapted Francisella is unknown. As a foundation for our goal of identifying F. tularensis outer membrane proteins which are expressed during mammalian infection, we first sought to identify in vitro cultivation conditions that induce the bacterium's infection-derived phenotype. We compared Francisella LVS grown in brain heart infusion broth (BHI; a standard microbiological medium rarely used in Francisella research) to that grown in Mueller-Hinton broth (MHB; the most widely used F. tularensis medium, used here as a negative control) and macrophages (a natural host cell, used here as a positive control). BHI- and macrophage-grown F. tularensis cells showed similar expression of MglA-dependent and MglA-independent proteins; expression of the MglA-dependent proteins was repressed by the supraphysiological levels of free amino acids present in MHB. We observed that during macrophage infection, protein expression by intracellular bacteria differed from that by extracellular bacteria; BHI-grown bacteria mirrored the latter, while MHB-grown bacteria resembled neither. Naïve macrophages responding to BHI- and macrophage-grown bacteria produced markedly lower levels of proinflammatory mediators than those in cells exposed to MHB-grown bacteria. In contrast to MHB-grown bacteria, BHI-grown bacteria showed minimal delay during intracellular replication. Cumulatively, our findings provide compelling evidence that growth in BHI yields bacteria which recapitulate the phenotype of Francisella organisms that have emerged from macrophages.

* Corresponding author. Mailing address: Center for Immunology and Microbial Disease, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208. Phone: (518) 262-2338. Fax: (518) 262-6161. E-mail: Hazlett{at}mail.amc.edu

{triangledown} Published ahead of print on 21 July 2008.

{dagger} Supplemental material for this article may be found at http://iai.asm.org/.

Editor: W. A. Petri, Jr.

Infection and Immunity, October 2008, p. 4479-4488, Vol. 76, No. 10
0019-9567/08/$08.00+0     doi:10.1128/IAI.00610-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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